This invention is directed to a reagent system for the determination of creatinine.
Creatinine, 1-methylglycocyamidine, C.sub.4 H.sub.7 ON.sub.3, is the end product of creatinine metabolism. It is one of the non-protein constituents of blood. Creatinine is a normal alkaline constituent of urine and blood. In humans, about 0.02 gm/kg. of body weight is excreted by the kidneys per day. Increased quantities of creatinine in the body fluids is generally an indication of renal malfunction.
There are several different methods for body fluid creatinine determination. Most of these methods are based on the picrate reaction with creatinine under alkaline conditions, as described by Jaffe M. Jaffe, Z. Physiol., Chem., 10(1886)391. The creatinine-picrate complex absorbs strongly at 520 nm, and its presence can be measured calorimetrically. The concentration of creatinine can thus be determined either by measuring the final absorbance at 520 nm, or by measuring the rate of change of absorbance at 520 nm. The latter is known as the rate Jaffe method.
A number of substances in body fluids are known to interfere with the Jaffe reaction, and increase the color yield. These substances include, for example, glucose, proteins, acetoacetate and pyruvate. Therefore, the Jaffe reaction suffers from lack of specificity. Attempts have been made to separate the creatinine from the interfering substances, proteins, carbohydrates, etc.), such as by means of ion exchange chromatography. But this method is time consuming, requires highly skilled operators, and is not suitable for routine analysis of a large number of samples.
The current practice is to use borates, and organic sulfates, to eliminate non-specific color formation due to interference by the reaction of chromogen-forming substances (other than creatinine) with picrate in alkaline conditions. This method enables creatinine determination without prior deproteinization. For example, proteins combine with sodium dodecyl sulfate (SDS) to form very stable complexes with a large number of negatively charged sulfate groups. Borates form negatively charged complexes with carbohydrates under alkaline conditions. Carbohydrates are neutral. One explanation of the efficacy of SDS and borate is that the negatively charged complexes and the picrate ions repel each other and are thus prevented from reacting. In this way nonspecific color formation in the Jaffe reaction can be reduced. Also, the presence of SDS allows the preparation of a reaction mixture blank, since acidification to eliminate the creatinine color does not precipitate the negative SDS-protein complexes. This allows the measurement of the blank absorbance. It was reported that the results obtained by using the SDS-borate reagents correlate well with those obtained with a method involving isolation of creatinine with a chromatographic procedure.
The pH of the reaction mixture also affects the color developed. The higher the pH, the faster the rate of color formation, and the stronger the final color. Current practice is to incorporate a pH buffer, such as a phosphate buffer, in the alkaline SDS-borate reagent This maintains the pH within a predetermined narrow range, such as a final reaction mixture pH of about 12 to 13.+-.0.2. The borate also acts as a pH buffer.
In modern automatic analyzers, reagents in substantial quantities are often stored on-board the equipment. Thus multiple samples can be analyzed before refilling is necessary. However, in clinical practice, many reagents are unstable at room temperatures, and require refrigeration. Therefore, certain modern automat-c analyzers are equipped with refrigerated reagent chambers. Therein lies the problem solved by the present invention.
Because of the relatively labile nature of the picrate ion in alkaline conditions, generally a liquid creatinine reagent based on the Jaffe reaction comprises two components. The first component, a picric acid solution, comprises picric acid and deionized water. The second component, an alkaline buffer, can comprise sodium dodecyl sulfate and sodium tetraborate for reducing interference by proteins and carbohydrates, respectively, sodium phosphate as pH buffer, sodium hydroxide to provide alkalinity, polypropylene glycol for foam control, and deionized water. The dynamic range of a creatinine reagent system is generally defined as the sample creatinine concentration range within which linear extrapolation of results is acceptable. For a creatinine reagent system having a dynamic range of 0.3 to 25 mg/dl creatinine, the following reagent system can be used: (a) picric acid component: 50 mM picric acid; (b) alkaline buffer: pH 13.1. The components are mixed in a proportion of about 1:4 picric acid: alkaline buffer) to form an assay ready reagent. The sample and the assay ready reagent is mixed in a proportion of about 1:5 sample:assay ready reagent). For the purpose of facilitating discussion, similar concentrations, ranges, and proportions are used throughout this application.
However, it was found that the active ingredients in the two separate components can precipitate out of solution at low temperatures For example, for a picric acid solution with a picric acid concentration of 50 mM or more, crystal formation occurs around 10.degree. C. As for the alkaline buffer, sodium dodecyl sulfate at a concentration of 1 5 wt. percent (in grams per deciliter) or more precipitates out at temperatures less than 18.degree. C. Other solutes such as sodium phosphate and sodium tetraborate also precipitate at near freezing temperatures.
A major problem with such precipitates is that they can interfere with reagent metering in the automatic analyzer. The precipitates in worst cases can clog up the micropipets, and generally interfere with the accurate metering of the liquid components. Thus assay reproducibility becomes a problem. One solution to the problem is of course to equip the analyzer with multiple reagent chambers, some refrigerated and some maintained at room temperature. But this is not satisfactory, as it involves more complicated engineering and thus higher costs.
What is needed is a reagent for the determination of creatinine, having components which do not have precipitation problems at low temperatures.